Humoral hypercalcaemia of "benignancy".
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Adolescent↗
Biomedical subjects
L J Deftos
Publications and source records attributed to L J Deftos.
Expression of parathyroid hormone-related peptide and its receptor messenger ribonucleic acid in human amnion and chorion-decidua: implications for secretion and function.
OBJECTIVE: Our purpose was to define the location and packaging of parathyroid hormone-related peptide in amnion-chorion and the potential target tissues for its action in fetal membranes. STUDY DESIGN: We studied fetal membranes by use of light microscopic immunocytochemistry with three monoclonal antibodies against distinct regions of the parathyroid hormone-related peptide molecule. For electron microscopy immunogold analysis with a monoclonal antibody specific to the 109-141 fragment was used to observe parathyroid hormone-related peptide intracellularly in amnion membrane and in the chorion layers. Multiplex reverse transcriptase-polymerase chain reaction with Southern blotting was used to identify parathyroid hormone/parathyroid hormone-related peptide receptor and control messenger ribonucleic acids in amnion and chorion-decidua. RESULTS: All monoclonal antibodies revealed immunoreactive parathyroid hormone-related peptide in the amniotic epithelial cells and in some fibroblast-like cells embedded in the extracellular matrix of the amnion. Parathyroid hormone-related peptide was also found in the chorion in fibroblast and trophoblast layers and in decidua. Ultrastructurally immunogold particles were evenly distributed throughout the amniotic epithelial cells and were present in apical microvilli and near the basal membranes. Electron microscopy studies of the chorion cytotrophoblast also showed freely dispersed immunogold particles of parathyroid hormone-related peptide with no packaging in secretory granules. Low to undetectable levels of parathyroid hormone/parathyroid hormone-related peptide receptor messenger ribonucleic acid were found in amnion tissue, whereas abundant receptor messenger ribonucleic acid was found in chorion-decidua. CONCLUSIONS: These results suggest the presence of a parathyroid hormone-related peptide paracrine system within the human fetal membranes.
Amnion↗
Immunohistochemical localization of parathyroid hormone-related protein in prostatic intraepithelial neoplasia.
Parathyroid hormone-related protein (PTHrP) is a regulatory protein hormone that has been associated with normal fetal growth and differentiation as well as fetal calcium regulation. Parathyroid hormone-related protein has been implicated in a variety of carcinomas as a major factor in the development of humoral hypercalcemia of malignancy and may also play a role as an autocrine growth factor. In a previous immunohistochemical study we found that all prostatic adenocarcinomas (CAP) express PTHrP. In the current study, we evaluated PTHrP in prostate intraepithelial neoplasia (PIN) in radical prostatectomy specimens. A validated mouse monoclonal antibody, 9H7, raised against fragment 109-141 of the carboxy-terminus of PTHrP was used for immunostaining. The results generally showed negative to weak staining of normal and hyperplastic tissue and strong staining in PIN. The staining intensity was further evaluated by computer based image analysis. The relative optical density in PIN (9.24 +/- 9.05) was significantly (P = .008) higher than that in normal gland (.00 +/- 3.6). These findings suggest that PTHrP may be involved in the pathogenesis of prostatic dysplasia, and its immunohistochemical evaluation may have diagnostic use in the evaluation of PIN.
Adenocarcinoma↗
Predicting PTH pulses and patterns in osteoporosis.
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Humans↗
Identification and characterization of a neuroendocrine-specific 5'-regulatory region of the human chromogranin A gene.
Chromogranin A (CgA) expression is specific to cells of endocrine and neuroendocrine (NE) tissues. Our transfection studies with CgA have identified two DNA regions 5' of the transcription start site that regulate CgA gene transcription: a distal regulatory region (DRR) located between -726 and -455, and a proximal regulatory region (PRR) between -60 and -26. In studies of the DRR using four human NE and six human non-NE cell lines, we demonstrated enhanced transcription of DRR-containing CgA-GH plasmids by the NE cells as a group compared to the non-NE cells. DNase I footprinting identified a protected area in the DRR from -570 to -555 base pairs (bp) composed of the sequence TAATGATGACTAAACA. Centered in this sequence is the simian virus 40 version of the activator protein-1-binding site, TGACTAA. Electrophoretic mobility shift assays (EMSAs) with an oligonucleotide containing the 27 bp of the DRR between -576 and -550, which we refer to as the distal regulatory element (DRE), produced a specific complex with the NE BEN and non-NE COS-1 cell nuclear extracts. The addition of c-Jun and c-Fos antibodies produced strong supershifts of the complex generated by COS-1 extract, but very weak supershifts of the complex formed by BEN extract. These EMSA studies suggest that NE cells such as BEN contain unique nuclear factors distinguishable from activator protein-1 that interact with the DRE. The enhancer effect of the 271-bp DRR could be replaced by the 27-bp DRE in both CgA and calcitonin promoter constructs in BEN cells. Replacement of the DRR with the DRE resulted in a further increase in expression from these plasmids, suggesting the presence of suppressor sequences in the DRR. In transfection studies of the PRR, deletion of its cAMP response element (CRE) dramatically lowered transcription. In addition to demonstrating that its CRE can bind CRE-binding protein, EMSAs with the PRR demonstrated that an intervening sequence between the CRE and the TATA box formed a complex with BEN cell nuclear extract. Our studies demonstrate that both the PRR and DRR are important for high level transcription of the CgA gene in NE cells. The presence of both distal and proximal 5'-regulatory regions in the human CgA gene indicates a complex mechanism of transcriptional regulation. Although the PRR is important for the formation of a functional transcription complex at the TATA region, the DRR is important for the enhancement of CgA gene expression in NE cells.
Base Sequence↗
Parathyroid hormone-related protein expression in human gastric adenocarcinomas not associated with hypercalcemia.
OBJECTIVE: Our evaluation of a patient with a parathyroid hormone-related protein (PTHrP)-producing gastric adenocarcinoma and hypercalcemia prompted us to study the expression of PTHrP in 13 additional patients with gastric cancer and in 10 control cases. Our objective was to investigate by immunohistology the expression of PTHrP in gastric cancer. METHODS: Immunohistology studies were conducted with two murine monoclonal antibodies to synthetic peptides of human PTHrP, 9H7, and 8B12. The 9H7 antibody was raised to the carboxy-terminal amino acid fragment (109-141) of PTHrP, and the 8B12 antibody was raised to the amino-terminal amino acid fragment (1-34) of PTHrP. RESULTS: Paraffin-embedded tumor specimens from 13 of 14 cases of gastric adenocarcinoma stained positively with the antibody to the carboxy terminus of the PTHrP peptide, but none stained positively with the antibody directed against the amino terminus. None of 10 control cases stained positively with either antibody. The staining was predominately evident in the cytoplasm of the tumor cells. Except for the sentinel case, none of the other patients with gastric adenocarcinoma had hypercalcemia. Thus, gastric adenocarcinoma seems to rarely result in systemic hypercalcemia. CONCLUSIONS: Our studies demonstrated that abnormal PTHrP production can occur in malignant cells without producing hypercalcemia. PTHrP may play a role in the pathogenesis of gastric adenocarcinoma that is independent of its hypercalcemic effects. PTHrP measurements may be clinically valuable in patients with cancer who are not hypercalcemic.
Adenocarcinoma↗
Parathyroid hormone-like protein: alternative messenger RNA splicing pathways in human cancer cell lines.
Parathyroid hormone-like protein (PLP) is expressed in a wide variety of cancers and exerts diverse biological effects in addition to hypercalcemia. We studied the expression of the gene for PLP in cancer cell lines derived from different tissues that produce PLP. We used the polymerase chain reaction to evaluate the PLP mRNA species produced in these various cell lines by differential transcription initiation and alternative splicing pathways. A series of exon-specific oligonucleotide primers that hybridize to DNA sequences adjacent to exon-intron splice junctions throughout the PLP gene were designed. These primers were used to evaluate steady state levels of PLP mRNA species. Our analysis with promoter-specific primers demonstrated expression from all three putative transcription start sites of PLP, designated P1, P2, and P3. P2-initiated transcripts were present in all of the cell lines, whereas the presence of P1- and P3-initiated messages were cell line specific. Our analysis with carboxy-terminal coding-specific primers demonstrated the utilization of the alternative splicing pathways that produce all three mature PLP polypeptides, PLP-139, -1-173, and -1-141. The 1-139 mRNA species was found in all of the cell lines, whereas the 1-141 and 1-173 mRNA species were cell line specific. These studies demonstrate the prevalence of the P2-initiated mRNA and the PLP1-139 alternative splicing pathway in the tumor cell lines studied and suggest that other pathways of PLP gene expression may be regulated in a cell line-dependent manner. The particular form of PLP expressed by a given cell can influence its biological effects.
DNA Primers↗
Islet amyloid polypeptide (amylin) increases the renal excretion of calcium in the conscious dog.
Islet amyloid polypeptide (IAPP) is a member of the calcitonin/CGRP family and has been isolated from the beta-cell of pancreatic islets. Recent evidence suggests that this peptide may be involved in calcium metabolism in that its administration resulted in lowering of serum calcium levels. To determine the mechanism of IAPP-induced hypocalcemia, the peptide was infused at 50 pmol/min/kg for 90 minutes in conscious male mongrel dogs. Infusion of the peptide resulted in a modest decline in the total serum calcium concentration (10.4 +/- 0.2 to 9.4 +/- 0.2 mg/dl; P < 0.05) and a concomitant increase in urinary calcium excretion (3.6 +/- 0.6 to 6.9 +/- 2.0 mg/dl; P < 0.01). Based on an extracellular volume of 7 liter in a 28 kg dog, the total decrement in calcium due to IAPP was 41.3 +/- 2.4 mg, whereas the total increase in urinary calcium was 3.2 +/- 0.7 mg. There were no detectable changes in calcitonin. We conclude that IAPP lowers serum calcium and increases the renal excretion of calcium independently of calcitonin. However, the calciuria can only account for a small component of the hypocalcemic effect and therefore, an additional calcium lowering effect of IAPP exits.
Amyloid↗
Parathyroid hormone-related protein is expressed by prostatic neuroendocrine cells.
OBJECTIVE: Parathyroid hormone-related protein (PTHrP) is a regulatory peptide that has been associated with normal fetal growth and differentiation as well as the regulation of fetal calcium. In a variety of cancers, PTHrP has been implicated in the humoral hypercalcemia of malignancy. Recently, we demonstrated that all prostatic adenocarcinomas express PTHrP. In the present study, the localization of PTHrP and its mRNA in nonmalignant prostate tissue was assessed. METHODS: Formalin-fixed, paraffin-embedded prostatic tissues from 23 patients were evaluated. Immunocytochemistry (ICC) was performed by the streptavidin-peroxidase enzyme conjugate method using a monoclonal antibody, 8B12, generated against fragment 1-34 of the amino-terminal end of PTHrP. Nonradioactive in situ hybridization was carried out using a digoxigenin labeled single-stranded cDNA probe complementary to the sequence coding for PTHrP(15-120). RESULTS: PTHrP immunoreactivity was observed in the cytosol of a few epithelial cells. Double labeling and serial section ICC with 8B12 and a monoclonal antibody to chromogranin A (a generic neuroendocrine [NE] marker) revealed that PTHrP was present in a subpopulation of prostatic NE cells. In situ hybridization of mirror image sections demonstrated positive signals in prostatic NE cells in complete accordance with the ICC findings. CONCLUSIONS: The localization and production of PTHrP in prostatic NE cells suggest that this polypeptide may act in an endocrine-paracrine fashion involved in the prostatic growth and differentiation as well as the regulation of calcium in semen and seminal fluid.
Gene Expression↗
Parathyroid hormone-related protein: a potential autocrine growth regulator in human prostate cancer cell lines.
OBJECTIVE: We recently demonstrated that parathyroid hormone-related protein (PTHrP) is widely expressed by human prostate cancer tissue, suggesting that PTHrP might be involved in the growth and development of prostate cancer. To study this further, the production of PTHrP and its biologic effect were investigated using human prostate cancer cell lines. METHODS: The cell lines used were one androgen-dependent cell line, LNCaP, and two androgen-independent cell lines, PC-3 and DU-145. PTHrP secreted by cancer cells was measured by radioimmunoassay. The effect of PTHrP on DNA synthesis in these cells was determined by thymidine incorporation assay. RESULTS: All cell lines secreted immunodetectable levels of PTHrP in the culture-conditioned media. PC-3 cells secreted significantly higher amounts than the other two cell lines. A synthetic peptide, PTHrP(1-34), stimulated thymidine uptake in PC-3 and DU-145 cells more than threefold the control under serum-free and steroid-free conditions, whereas LNCaP was not affected. However, in the presence of dihydrotestosterone, DNA synthesis of LNCaP cells was stimulated by PTHrP in a dose-dependent manner. Additionally, this PTHrP-induced DNA synthesis was completely neutralized by a validated mouse monoclonal antibody (8B12) raised against PTHrP(1-34). CONCLUSIONS: Our data suggest that PTHrP may play a significant role in the growth of prostate cancer by acting locally in an autocrine fashion.
DNA, Neoplasm↗
Alveolar epithelial cells express and secrete parathyroid hormone-related protein.
Parathyroid hormone-related protein (PTHrP) was discovered as a hypercalcemia-inducing product of malignant cells and has since been demonstrated to be a product of many tissues. Although it is robustly expressed in fetal lung, PTHrP expression has not been assigned to alveolar epithelial cells in adult lung. We have shown that PTHrP is expressed in the adult rat lung and by cultured rat alveolar type II epithelial cells with sensitive and specific immunoassays and immunohistochemical techniques. Immunoassay of cell extracts demonstrated that freshly isolated type II cells contained PTHrP (136 pg/10(7) cells), whereas freshly isolated alveolar macrophages and cultured macrophages did not express PTHrP. Cultured type II cells secreted PTHrP into medium, 202 +/- 11 fg PTHrP/micrograms cell protein in 24 h. Basal secretion remained stable up to 7 days in culture. Treatment with phorbol myristate acetate or 1-oleoyl-2-acetyl-sn-glycerol produced a dose-related, 2- to 4-fold increase in PTHrP secretion. However, forskolin, ionomycin, ATP, phenylephrine, capsaicin, and bradykinin had no effect. Thus, PTHrP secretion appeared to be regulated by a protein kinase C-dependent pathway. PTHrP could also be demonstrated in pulmonary lavage fluid. Although the function of PTHrP in the adult lung is unknown, it could involve control of cell growth and differentiation or control of surfactant lipid secretion. Further studies are necessary to elucidate the function of PTHrP in the lung.
Animals↗
Parathyroid hormone-related protein in the cardiovascular system.
PTH-related protein (PTHrP) is expressed in a stretch-responsive manner in several types of smooth muscle. We previously demonstrated the production of PTHrP in adult rat heart muscle. In this study, we demonstrate the production of PTHrP in the cardiovascular systems of several mammalian species, including human. We demonstrate PTHrP by immunohistology, using a panel of murine monoclonal antibodies to PTHrP epitopes that span the entire length of the human PTHrP amino acid sequence, quantitate the concentration of PTHrP in the rat cardiovascular system by region-specific RIAs, and measure the relative levels of PTHrP messenger RNA (mRNA) by competitive polymerase chain reaction. Immunohistology studies demonstrated the presence of PTHrP in the cardiovascular systems of humans, rats, pigs, and rabbits. The most robust expression was found in atria, followed by the large vessels, then ventricles. No difference was seen between the left and right sides of the heart. Double staining procedures revealed that PTHrP and atrial naturietic peptide were coexpressed in some cells. Using RIAs and polymerase chain reaction, we demonstrated that atria contained a higher concentration of PTHrP than ventricles and that the relative PTHrP concentrations correlated to its mRNA concentrations in these two tissues. The concentrations of PTHrP in the smooth muscle surrounding the aorta and vena cava were comparable to those in atria. However, in these large vessels, the higher PTHrP levels did not correspond to its mRNA levels. Whereas the immunoreactive concentrations of PTHrP were similar in the atria, aorta, and vena cava, the mRNA levels in the aorta and vena cava were 3-fold lower than those in the atria. Certain PTHrP epitopes appeared to be differentially expressed in specific cardiovascular tissues. A comparison of region-specific assays showed that immunoreactivity measured by immunoassays to PTHrP-(38-64) and PTHrP-(109-141) were 3- to 5-fold greater than that determined by an immunoassay to PTHrP-(1-34). Our observations demonstrate that the atria, aorta, and vena cava contain the greatest amounts of PTHrP in the cardiovascular system. The discrepancy between the concentrations of PTHrP and its mRNA present in the aorta and vena cava suggest that the two may be regulated differently in these tissues. The widespread distribution of PTHrP suggests an important function for the protein in the cardiovascular system, possibly functioning as the calcium counterpart for the atrial natriuretic-sodium regulatory axis.
Animals↗
The short-term effects of conjugated estrogen on bone turnover in older women.
Estrogen replacement therapy (ERT) prevents bone loss and fracture in early postmenopausal women, but its benefit for women over 70 yr of age has not been determined. We have examined the effect of a short course of ERT on biochemical markers of bone turnover in older women. Eleven women (mean age, 77 yr) were given conjugated estrogen (Premarin; 0.625 mg/day) for 6 weeks. Biochemical markers were measured on serum and urine collected at baseline (two samples), after 5 and 6 weeks of ERT, and 5 and 6 weeks post-ERT. Markers of bone formation were osteocalcin, bone alkaline phosphatase, and type I procollagen peptide. Markers of bone resorption were total urinary hydroxyproline, total and free pyridinoline and deoxypyridinoline cross-links, type I collagen cross-linked N-telopeptides, and serum C-terminal cross-linked telopeptide. Data were analyzed by repeated measures multivariate analysis of variance to estimate the overall effect of ERT on the biochemical markers. Markers of bone resorption decreased during ERT and returned to baseline after ERT (P < 0.05). Markers of bone formation declined less during ERT and continued to decline after ERT (P < 0.05). We conclude that ERT reduces bone turnover in older women and that markers of bone turnover may be useful in assessing the response to treatment in this age group.
Aged↗
Chromogranin A peptide-specific antisera and high-performance size exclusion chromatography demonstrate amino-terminal and carboxy-terminal fragments of the native molecule in human cell lines.
We have studied the pattern of amino-terminal and carboxy-terminal protein processing of chromogranin A (CgA) in five human cell lines, four derived from lung cancers: NCI-H478, NCI-H1011, NCI-H727, and BEN; and one derived from human medullary thyroid carcinoma: TT. This was accomplished by fractionation of cell extracts by high-performance size exclusion chromatography (HPSEC) and measurement of CgA in the fractions by radioimmunoassay (RIA). Three RIA's were used, one specific for the amino-terminus of CgA, one specific for the carboxy-terminal region, and a monoclonal antibody-based assay that recognizes only the native CgA molecule. We demonstrated the presence of different amino- and carboxy-terminal immunoreactive species of CgA in the different cell lines. The amino-terminal assay demonstrated distinct low-molecular-size species in the NCI-H478 and NCI-H1011 cell lines, and a similar peak in the TT cells. The amino-terminal assay did not recognize any distinct species in BEN and NCI-H727 cell lines. The carboxy-regional assay demonstrated distinct low-molecular-size species in the NCI-H478 and NCI-H101 cell lines and high-molecular-size species in the NCI-H727 and BEN cells. Our studies demonstrate with region-specific RIA's the presence of both amino- and carboxy- forms of CgA in human cells that secrete this protein. These results provide direct evidence that CgA-producing cells produce, probably through endoproteolytic processing of the native molecule, amino- and carboxy-terminal forms of the protein.
Amino Acid Sequence↗
Neoplastic hormone-producing cells of the placenta produce and secrete parathyroid hormone-related protein. Studies by immunohistology, immunoassay, and polymerase chain reaction.
BACKGROUND: Parathyroid hormone-related protein (PTHrP) was originally discovered as a product of tumors associated with hypercalcemia. Subsequent studies have demonstrated the widespread production of PTHrP in both fetal and adult tissues, among them the placenta, where it has been identified in amniotic epithelial cells and trophoblasts. However, studies of PTHrP production by the neoplastic placenta have not been reported. EXPERIMENTAL DESIGN: We have studied placental cells and tissues for PTHrP by immunohistology, immunoassay, and the polymerase chain reaction. We studied normal placenta, placenta neoplasia, and placental cell lines grown in culture. RESULTS: Our studies in abnormal placenta demonstrate that PTHrP is produced by chorioangioma, choriocarcinomas, and hydatidiform moles, indicating that PTHrP may be a marker for neoplasia of the placenta. Our studies in normal placenta demonstrate that PTHrP is produced by several placental cell types, including, syncytiotrophoblast, intermediate trophoblasts, cytotrophoblasts, and amniotic epithelium, suggesting an important role for this protein in several aspects of placental function. Cell lines derived from trophoblasts and amniotic epithelium also produced PTHrP and secreted it into culture medium. Polymerase chain reaction with PTHrP coding sequence primers demonstrated the appropriate polymerase chain reaction product in these placental cells. CONCLUSIONS: The production of PTHrP by several normal and abnormal placental cell types is evidence for an important function for PTHrP in normal and neoplastic placenta.
Cell Line↗
Immunohistochemical localization of parathyroid hormone-related protein in human prostate cancer.
Parathyroid hormone-related protein (PTHrP) is produced by a variety of malignant tumors and has been implicated as a major cause of humoral hypercalcemia of malignancy. Expression of PTHrP in prostate cancer tissue was studied immunohistochemically using 33 radical prostatectomy specimens from patients with clinically localized carcinoma of the prostate. None of these patients demonstrated hypercalcemia prior to the surgery. Acetone-methyl benzoate-xylene-processed, paraffin-embedded tissues were stained with a validated mouse monoclonal antibody to an amino acid fragment, PTHrP(109-141), using the streptavidin-peroxidase enzyme conjugate method. All cases (33 of 33; 100%) studied demonstrated some degree of immunoreactivity throughout the cytoplasm of the tumor cells, but immunostaining was absent from inflammatory and stromal cells. The intensity of the staining appeared to directly correlate with increasing tumor grade. The widespread immunohistochemical localization of PTHrP in carcinoma of the prostate suggests that PTHrP may play some local role in the growth of transformed cells in the prostate. Furthermore, overexpression of PTHrP may be a possible marker to evaluate the malignant potential of carcinoma of the prostate.
Aged↗
Vitamin D3 compounds regulate human immunodeficiency virus type 1 replication in U937 monoblastoid cells and in monocyte-derived macrophages.
We studied the effects of vitamin D3 compounds on the replication of human immunodeficiency virus type 1 (HIV-1) in the monoblastoid cell line U937 and in primary monocyte-derived macrophage cultures to understand how modulators of monocyte/macrophage effector function might affect the pathogenesis of HIV-1 infection. U937 cell cultures exposed to 1, alpha 25-dihydroxyvitamin D3 prior to HIV-1 infection showed enhanced virus replication that was apparently due to increased cellular resistance to viral cytopathic effects; a marked inhibition of virus replication was noted in cells exposed to 1 alpha,25-dihydroxyvitamin D3 subsequent to infection. Exposure of blood-derived monocyte/macrophages to vitamin D3 compounds prior to infection also affected virus growth; in most cases, substantial inhibition of HIV-1 replication was noted in vitamin D3-treated macrophage cultures. Our results demonstrate that vitamin D3 compounds with recognized abilities to induce cellular differentiation can modulate HIV-1 infection of human macrophages.
Calcifediol↗
Associations and dissociations between serum bone Gla protein and alkaline phosphatase in skeletal metabolism.
Serum bone Gla protein (BGP) and alkaline phosphatase (AP) activity were compared for the assessment of skeletal status in 11 normal pregnant women, 12 normal women on days 3 and 13 of the menstrual cycle, five postmenopausal women before and after 1 month of treatment with ethinyl estradiol (20 micrograms/day), five patients with cancer and hypercalcemia during treatment with calcitonin, and one patient with Paget disease during treatment with Plicamycin. BGP and AP correlated with each other only in the pregnant women. In all other circumstances, there was no correlation between these two serum osteoblast products. Furthermore, there were conditions in which the two measurements became discordant. These studies demonstrate that BGP and AP commonly are dissociated when used as measurements of skeletal status. Although both are osteoblast products, BGP and AP probably reflect different aspects of osteoblast differentiation and function.
Adult↗